The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated ion channel present in the apical surface of several epithelia including the airways, pancreas, kidney, sweat glands and gastrointestinal tract. CFTR is central to several disease pathologies including cystic fibrosis, polycystic kidney disease and enterotoxin-induced secretory diarrheas such as cholera. The long-term objective of this proposal is to use live cell fluorescence imaging to address key unresolved issues in the CFTR field. Specific Aim 1 investigates the structure of CFTR channels in live cells using single molecule fluorescence techniques. The hypotheses will be tested that PDZ domain interactions at the C-terminus of CFTR and activation of protein kinases modulate the oligomeric state of CFTR. These studies will yield insight into the structure of CFTR and possible mechanisms that regulate channel activity. Specific Aim 2 investigates CFTR trafficking at the apical plasma membrane of cells. CFTR will be directly labeled to visualize individual endocytic and recycling events with excellent spatial and temporal resolution. The role of protein kinase A and CFTR domain interactions in the itinerary of CFTR will be determined. Regulation of CFTR trafficking is one of the most controversial areas in the field and resolution of this issue is warranted. Specific Aim 3 will investigate whether CFTR is integrated into a cell surface protein complex with regulatory and structural proteins. The dynamics of interactions between CFTR and 22-adrenergic receptor (22AR), a key regulator of CFTR activity, will be probed by two color single particle tracking techniques. The hypothesis will be tested that CFTR and 22AR form transient, as opposed to stable, complexes to modulate CFTR activity. The proposed experiments will provide new insights into the structure, activity and regulation of CFTR activity that are not possible with alternative techniques. PUBLIC HEALTH RELEVANCE: The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel present in many tissues including the airways and digestive tract. CFTR activity is important in several diseases including cystic fibrosis, polycystic kidney disease and secretory diarrheas (e.g., cholera). The goal of this proposal is to use live cell microscopy to investigate unresolved issues in the field of CFTR research.